Mett19基因?qū)251膠質(zhì)瘤細(xì)胞增殖和凋亡的影響
發(fā)布時間：2018-10-21 14:44
【摘要】：膠質(zhì)瘤是臨床最常見的原發(fā)性中樞神經(jīng)系統(tǒng)惡性腫瘤,它本質(zhì)上是一種多基因異常疾病,通過一種或多種癌基因的過度表達(dá)、同時伴隨抑癌基因的突變?nèi)笔亩剐盘杺鲗?dǎo)通路異常,腫瘤細(xì)胞逃逸了正常生長調(diào)控機制,結(jié)果出現(xiàn)細(xì)胞的異常增殖、自主侵襲、血管增生等惡性表型。Mettl9基因是新發(fā)現(xiàn)的一個p53下游基因,在肺癌、小鼠胎肺和SD大鼠中樞神經(jīng)系統(tǒng)的發(fā)育過程中特異性表達(dá),且該基因表達(dá)信號的強弱與凋亡細(xì)胞的強弱成正相關(guān),其在胚胎發(fā)育過程中的作用很可能是通過參與細(xì)胞凋亡來實現(xiàn)的。因此,本研究中,我們觀察不同表達(dá)水平的Mett19基因?qū)δz質(zhì)瘤U251細(xì)胞增殖和凋亡的影響,初步探討及作用機制。 目的： 1.研究Mett19基因的不同表達(dá)水平對U251細(xì)胞生長的影響； 2.探討Mett19基因?qū)251生長的作用機制。 方法： 應(yīng)用RT-PCR方法,擴增出Mett19基因的cDNA,克隆至pGEM-T載體上并測序,再將該基因亞克隆至慢病毒載體pLenti6.3-MCS-IRES-EGFP,在293T細(xì)胞進(jìn)行病毒包裝,構(gòu)建Mettl9基因的慢病毒過表達(dá)載體；根據(jù)RNA干擾序列設(shè)計原則,設(shè)計RNA干擾靶點序列,合成含干擾序列的雙鏈DNA oligo,通過酶切連接構(gòu)建Mett19基因的RNAi干擾載體。實驗分為五組：正常培養(yǎng)細(xì)胞組,慢病毒空載體組(NC),慢病毒過表達(dá)載體組(Mett19組),干擾載體對照組(NC-RNAi組),Mett19干擾載體組(Mettl9-RNAi組)。將上述已經(jīng)構(gòu)建的各質(zhì)粒分別瞬時感染或轉(zhuǎn)染U251細(xì)胞,48h后,熒光顯微鏡觀察GFP綠色熒光表達(dá)情況,qRT-PCR檢測Mett19基因的表達(dá)水平,以此評價病毒感染或質(zhì)粒轉(zhuǎn)染后Mett19基因的表達(dá)效率；收集各組細(xì)胞,MTT方法檢測細(xì)胞活力,流式細(xì)胞術(shù)檢測細(xì)胞凋亡情況,qRT-PCR方法檢測凋亡相關(guān)因子Bcl-2, Bax mRNA的表達(dá)水平。結(jié)果： 1.Mettl9cDNA的RT-PCR擴增產(chǎn)物連接到pGEM(?)-T載體,測序結(jié)果向GenBank遞交獲得收錄號HQ898855； 2.成功構(gòu)建了Mett19基因的慢病毒過表達(dá)載體和RNAi干擾載體； 3.熒光顯微鏡下可見GFP綠色熒光的表達(dá)陽性率約為50%,感染Mett19慢病毒過表達(dá)質(zhì)粒,Mett19的表達(dá)水平明顯高于NC對照組)；而轉(zhuǎn)染Mettl9-RNAi組,Mett19的表達(dá)水平明顯低于NC-RNAi組； 4.MTT結(jié)果顯示,感染Mett19慢病毒過表達(dá)質(zhì)粒組,細(xì)胞增殖能力明顯降低；而轉(zhuǎn)染Mettl9-RNAi組,細(xì)胞增殖能力明顯增加； 5.流式細(xì)胞術(shù)結(jié)果顯示,感染Mett19慢病毒過表達(dá)質(zhì)粒組,細(xì)胞凋亡率明顯增加；而轉(zhuǎn)染Mettl9-RNAi組,細(xì)胞凋亡率明顯降低)； 6.qRT-PCR結(jié)果顯示,感染Mett19慢病毒過表達(dá)質(zhì)粒組,凋亡相關(guān)因子Bcl-2mlNA的表達(dá)水平降低,Bax mRNA的表達(dá)水平明顯升高,差異具有統(tǒng)計學(xué)意義；而轉(zhuǎn)染Mettl9-RNAi組,凋亡相關(guān)因子Bcl-2mRNA的表達(dá)水平升高,Bax mRNA的表達(dá)水平明顯降低,差異具有統(tǒng)計學(xué)意義。 結(jié)論：過表達(dá)Mett19基因抑制U251細(xì)胞增殖,促進(jìn)其凋亡,可能與Bcl-2mRNA表達(dá)水平下降,Bax mRNA表達(dá)水平升高有關(guān)。
[Abstract]:Glioma is the most common primary malignant tumor of the central nervous system. It is essentially a polygenic disorder, which is overexpressed by one or more oncogenes. At the same time, with the mutation and deletion of tumor suppressor gene, the signal transduction pathway is abnormal, tumor cells escape the normal growth regulation mechanism, resulting in abnormal cell proliferation and spontaneous invasion. Angiogenesis and other malignant phenotypes. Mettl9 gene is a newly discovered p53 downstream gene that is specifically expressed in the central nervous system of lung cancer, mouse fetal lung and SD rats. Moreover, the intensity of the gene expression signal was positively correlated with that of apoptotic cells, and its role in embryonic development was probably realized by taking part in apoptosis. Therefore, in this study, we observed the effect of Mett19 gene at different levels on the proliferation and apoptosis of U251 glioma cells. Objective: 1. To study the effects of different expression levels of Mett19 gene on the growth of U251 cells. 2. To explore the mechanism of Mett19 gene acting on U251 growth. Methods: the cDNA, of Mett19 gene was amplified by RT-PCR and sequenced into the pGEM-T vector. Then it was subcloned into the lentivirus vector pLenti6.3-MCS-IRES-EGFP, for viral packaging in 293T cells to construct the lentivirus overexpression vector of Mettl9 gene. According to the principle of RNA interference sequence design, the RNA interference target sequence was designed, and the double-stranded DNA oligo, containing interference sequence was synthesized and ligated to construct the RNAi interference vector of Mett19 gene by restriction endonuclease ligation. The experiment was divided into five groups: normal culture cell group, lentivirus empty vector group, (NC), lentivirus overexpression vector group (Mett19 group), interference vector control group (NC-RNAi group), Mett19 interference vector group (Mettl9-RNAi group). The constructed plasmids were transiently infected or transfected into U251 cells respectively. After 48 hours, the green fluorescence expression of GFP was observed by fluorescence microscope and the expression level of Mett19 gene was detected by qRT-PCR to evaluate the expression efficiency of Mett19 gene after virus infection or plasmid transfection. The cell viability was detected by MTT, apoptosis was detected by flow cytometry, and the expression of apoptosis-related factor (Bcl-2, Bax mRNA) was detected by qRT-PCR. Results: the RT-PCR amplification product of 1.Mettl9cDNA was ligated to pGEM (?) -T vector, and the result of sequencing was submitted to GenBank to obtain HQ898855; 2. The lentivirus overexpression vector and RNAi interference vector of Mett19 gene were successfully constructed. Under fluorescence microscope, the positive rate of GFP green fluorescence was about 50%, the expression level of Mett19 in infected Mett19 lentivirus overexpression plasmid was significantly higher than that in NC control group, but the expression level of Mett19 in Mettl9-RNAi group was significantly lower than that in NC-RNAi group. The results of 4.MTT showed that the ability of cell proliferation was significantly decreased in the group infected with Mett19 lentivirus overexpression plasmid, while in the group transfected with Mettl9-RNAi, the ability of cell proliferation was significantly increased. The results of flow cytometry showed that the rate of apoptosis was significantly increased in the group infected with Mett19 lentivirus overexpression plasmid, while the rate of apoptosis was significantly decreased in the group transfected with Mettl9-RNAi. The results of 6.qRT-PCR showed that the group infected with Mett19 lentivirus overexpression plasmid group. The expression level of apoptosis-related factor (Bcl-2mlNA) decreased significantly, and the expression level of, Bax mRNA increased significantly (P < 0.05), while in Mettl9-RNAi transfected group, the expression level of Bcl-2mRNA increased and the expression of, Bax mRNA decreased significantly. The difference is statistically significant. Conclusion: overexpression of Mett19 gene inhibits the proliferation of U251 cells and promotes its apoptosis, which may be related to the decrease of Bcl-2mRNA expression level and the increase of, Bax mRNA expression level.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R739.41
本文編號：2285416
[Abstract]:Glioma is the most common primary malignant tumor of the central nervous system. It is essentially a polygenic disorder, which is overexpressed by one or more oncogenes. At the same time, with the mutation and deletion of tumor suppressor gene, the signal transduction pathway is abnormal, tumor cells escape the normal growth regulation mechanism, resulting in abnormal cell proliferation and spontaneous invasion. Angiogenesis and other malignant phenotypes. Mettl9 gene is a newly discovered p53 downstream gene that is specifically expressed in the central nervous system of lung cancer, mouse fetal lung and SD rats. Moreover, the intensity of the gene expression signal was positively correlated with that of apoptotic cells, and its role in embryonic development was probably realized by taking part in apoptosis. Therefore, in this study, we observed the effect of Mett19 gene at different levels on the proliferation and apoptosis of U251 glioma cells. Objective: 1. To study the effects of different expression levels of Mett19 gene on the growth of U251 cells. 2. To explore the mechanism of Mett19 gene acting on U251 growth. Methods: the cDNA, of Mett19 gene was amplified by RT-PCR and sequenced into the pGEM-T vector. Then it was subcloned into the lentivirus vector pLenti6.3-MCS-IRES-EGFP, for viral packaging in 293T cells to construct the lentivirus overexpression vector of Mettl9 gene. According to the principle of RNA interference sequence design, the RNA interference target sequence was designed, and the double-stranded DNA oligo, containing interference sequence was synthesized and ligated to construct the RNAi interference vector of Mett19 gene by restriction endonuclease ligation. The experiment was divided into five groups: normal culture cell group, lentivirus empty vector group, (NC), lentivirus overexpression vector group (Mett19 group), interference vector control group (NC-RNAi group), Mett19 interference vector group (Mettl9-RNAi group). The constructed plasmids were transiently infected or transfected into U251 cells respectively. After 48 hours, the green fluorescence expression of GFP was observed by fluorescence microscope and the expression level of Mett19 gene was detected by qRT-PCR to evaluate the expression efficiency of Mett19 gene after virus infection or plasmid transfection. The cell viability was detected by MTT, apoptosis was detected by flow cytometry, and the expression of apoptosis-related factor (Bcl-2, Bax mRNA) was detected by qRT-PCR. Results: the RT-PCR amplification product of 1.Mettl9cDNA was ligated to pGEM (?) -T vector, and the result of sequencing was submitted to GenBank to obtain HQ898855; 2. The lentivirus overexpression vector and RNAi interference vector of Mett19 gene were successfully constructed. Under fluorescence microscope, the positive rate of GFP green fluorescence was about 50%, the expression level of Mett19 in infected Mett19 lentivirus overexpression plasmid was significantly higher than that in NC control group, but the expression level of Mett19 in Mettl9-RNAi group was significantly lower than that in NC-RNAi group. The results of 4.MTT showed that the ability of cell proliferation was significantly decreased in the group infected with Mett19 lentivirus overexpression plasmid, while in the group transfected with Mettl9-RNAi, the ability of cell proliferation was significantly increased. The results of flow cytometry showed that the rate of apoptosis was significantly increased in the group infected with Mett19 lentivirus overexpression plasmid, while the rate of apoptosis was significantly decreased in the group transfected with Mettl9-RNAi. The results of 6.qRT-PCR showed that the group infected with Mett19 lentivirus overexpression plasmid group. The expression level of apoptosis-related factor (Bcl-2mlNA) decreased significantly, and the expression level of, Bax mRNA increased significantly (P < 0.05), while in Mettl9-RNAi transfected group, the expression level of Bcl-2mRNA increased and the expression of, Bax mRNA decreased significantly. The difference is statistically significant. Conclusion: overexpression of Mett19 gene inhibits the proliferation of U251 cells and promotes its apoptosis, which may be related to the decrease of Bcl-2mRNA expression level and the increase of, Bax mRNA expression level.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R739.41
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